Inhibition of human cancer cell growth by inducible expression of human ribonucleotide reductase antisense cDNA

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis an d repair. The enzyme consists of two dissimilar subunits, M1 and M2, It is known that the M2 subunit plays a role in tumorgenicity and metastasis, In this study, we transfected human oropharyngeal KB cancer cells with human R R M1 and M2 antisense cDNA expressed by an inducible vector system. The tra nsfectants were double-selected with hygromycin and G418, The clones, desig nated KB-M1AS, KB-M2AS and KB-CAT, represented transfectant clones that con tained M1 antisense cDNA, M2 antisense cDNA, and a CAT reporter gene, respe ctively. In a colony-forming assay, colony formation for the KB-M2AS clone decreased approximately 50% when M2 antisense mRNA expression was induced b y isopropylthiogalactose (IPTG), However, the KB-M1AS clone revealed no sig nificant inhibition under IPTG induction. RR enzyme activity, as measured b y (CDP)-C-14 reduction assay, revealed a 30% decrease in the IPTG-induced K B-M2AS clone relative to non-IPTG-induced samples at 144 hours. As shown by Northern blot, expression of the M2 antisense mRNA showed peaks at 48 hour s and 144 hours after induction by IPTG, M2 antisense mRNA expression induc ed by IPTG was 33-fold greater than the uninduced control at 144 hours, Wes tern blot analysis showed that the M2 subunit protein level decreased in th e KB-M2AS clone beginning at 72 hours after induction and continued to decr ease to 50% of the uninduced control at 144 hours, then showed a slight rec overy at 168 hours. In conclusion, M2 antisense mRNA expression by an induc ible system can effectively decrease RR M2 protein expression, reduce enzym e activity, and inhibit growth. Furthermore, this approach can be employed in future antisense investigations.